Increased production of IL-15 protein in adult coeliac disease
patients
Escudero-Hernández C.1; Vallejo-Díez S. 1; Martínez-Abad B. 1; Montalvillo E. 1; Marugán M. 4; Fernández-Salazar L. 3; Garrote J.A.2; Arranz E. 1
1. Mucosal Immunology Lab, IBGM, University of Valladolid-CSIC, Spain 2. Molecular Genetics, Hospital Universitario Rio Hortega, Valladolid, Spain.
3. Gastroenterology Service, Complejo Hospitalario de León, Spain 4. Paediatrics Service, Hospital Clínico Universitario Valladolid, Spain.
Corresponding author : earranz@med.uva.es
Introduction:
Patients with coeliac disease (CD) may have an increased expression of the specific receptor of interleukin (IL)-15 (IL-15Ra) but not in healthy individuals. (Bernardo D. et al. 2008) Furthermore, gluten
ingestion in both groups increases the expression of IL-15. Therefore, the study of this cytokine and its receptor could be of great interest in order to obtain a better understanding of CD pathogenesis.
Patients and methods:
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Western blot has been used to determine the expression of IL-15 and IL-15Ra using b-actin as a reference. Samples: duodenal biopsies from coeliac patients, CD (n=17: 6 children, 7 adults, 4 non good responders to gluten-free diet (GFD), one of which is refractory CD patient), non-coeliac pathologic controls, Pc (n=10) and apparently healthy individuals, Hc (n=10). Versadoc and Quantity One
sorftwares (BioRad) were used to develop and quantify, respectively.
Quantitative PCR using the same samples as western blot was performed for analysis of IL-15Ra, IL-15, STAT3, STAT5A, STAT5B, IL-21 and IFN-g. GADPH was quantified as a reference.
We used also flow cytometry to study cell level expression of IL-15 and IL-15Ra, extracellular and intracellular, using duodenal biopsies from coeliac patients (n=5), non-coeliac pathologic controls
(n=4) and apparently healthy individuals (n=5) Intraepithelial lympocytes (IELs) and lamina propia lymphocytes (LPLs) were separated using 1M DTT , 1M EDTA and incubated in completed culture
medium for 1h at 37ºC. Lamina propia was then isolated using collagenase (2h at 37ºC)
Statistical analysis were performed using the non-parametric Mann-Whitney’s test and t-test.
Results:
1.A
Western blot: IL-15Ra analisys shows two bands (approx. molecular weights of 40 and 48KDa) whereas IL15 shows five bands: approx. 14KDa (IL-15), 17 KDa (Short signal peptide IL-15) and 20KDa (long signal
peptide IL-15), but also 40 KDa and 48KDa bands. (Fig. 1)
The appearance of the two high molecular bands in IL-15 and IL-15Ra western blots indicate that both
proteins are bound together. The different molecular weight could be due to a different N-glycosilation
process inside cells; being the high molecular weight band the one secreted in the cellular membrane.
Densitometric analysis: some degree of increase of IL-15 secretion (48KDa band) in adult coeliac patients
when compared to healthy controls (p=0.184) and paediatric CD patients (p=0.174) was observed.
Surprisingly, levels of secreted IL-15 are significantly decreased in non-responding CD patients (p<0.1)
Finally, IL-15Ra western blot did not show any significant variation, though a tendency can be appreciated.
(Fig.2)
1.00
0.80
0.60
0.40
0.20
0.00
48KDa
Hc
Pc
Child CD
40KDa
Adult CD
Non-resp CD
IL-21 expression pattern do not display any possible
increment. IFN-g was analyzed because of its ability of
inducing IL-15, but also as an imflammatory marker. Its
relative quantification is consistent with previous studies.
(Fig.5)
Pc
child CD
adult CD
48 and
40KDa
IL-15
bands
Figure 1: Representative IL-15Ra (A) and IL-15 (B) western blots. IL15Ra western blot shows two bands (approx. molecular weights of
40 and 48KDa) IL-15 western blot show five bands: approx. 14KDa
(IL-15), 17 KDa (Short signal peptide IL-15) and 20KDa (long signal
peptide IL-15), but also 40 KDa and 48KDa bands. Healthy controls,
Hc; pathologic controls, Pc; coeliac disease, CD.
3
2.00
1.50
1.00
0.50
0.00
48KDa
Hc
Pc
40KDa
LSP IL15
Child CD
Adult CD
SSP IL15
IL15
Non-resp CD
Figure 2 : Graphic representation of densitometric analysis of IL-15Ra (A) and IL-15 (B) western blot and median
values for each group. Hc, healthy individuals; Pc, pathological controls; CD, coeliac disease patients; Non-resp.
GFD, non-responders to gluten-free diet.
Quantitative PCR: IL-15 expression was compared to STAT3,
STAT5A and STAT5B, as molecules included in the JAK/STAT
signaling pathway through IL-15Ra activation by IL-15. (Fig.4)
While healthy control expression show lower levels in each
molecule, pathological controls and CD patients show higher
expression of STAT5A and STAT5B. STAT3 is the only molecule
where paediatric CD levels are similar to healthy controls. On
the other hand, non-responding GFD CD patients show similar
levels of mRNA for IL-15, STAT3 and STAT5B, but STAT5A.
(Fig.4)
Pc
1.B
IL-15 western blot
Median values of the relative
quantification (arbitrary units)
Median values of the relative
quantification (arbitrary units)
2.B
IL-15Ra western blot
Pc
48KDa
40KDa
Flow cytometry: A set of IL-15Ra, IL-15, CD45 and CD3 or CD11c antibodies were used to assess the
amounts of IL-15 and IL-15Ra proteins in both duodenal intraepithelial and lamina propria lymphocytes
and dendritic cells. No significant results were obtained. Compared to western blot, flow cytometry does
not provide additional information.
2.A
Hc
4
Figure 3: Graphic representation of the relative quantification (2(DDCt) method) of IL-15Ra and IL-15 expression respect to healthy
controls by qPCR.
5
Figure 4: Relative quantification (2-(DDCt) method)
of IL-15, STAT3, STAT5A and STAT5B expression by
qPCR.
Figure 5: Relative quantification (2-(DDCt) method)
of IL-15, IL-21 and IFN-g expression by qPCR.
Conclusions:
IL-15 is tightly regulated at a post-transcriptional level. Because of that, IL-15 should be bore in mind together with its specific receptor IL-15Ra. Here we demonstrate that the IL-15/IL15Ra assosiation can be detected by western blot. Furthermore, soluble IL-15Ra (sIL-15Ra) could have been detected (approx. Molecular weight: 42KDa) (Duitman E. et al. 2008)
Although IL15 expression is not very reliable because of its strict post-transcriptional regulation, IL-15Ra expression shows the same patter as western blot for pathological controls, children
CD and adult CD patients. What is more, different expression patterns in other IL-15-related molecules could be important in different ages and/or stages of the disease (non-good
responders to GFD express less STAT5A). On the other hand, GFD non-responding patients do not reach the expected increased levels of IL-15 mRNA expressión or protein as well as IFN-g
mRNA when compared to the remainder CD groups.
References::
Bernardo D. et al. 2008 Clin. Exp. Immunol., 154: 64-73 “Higher constitutive IL-15Ra expression and lower IL-15 response threshold in coeliac disease patients”.
Duitman E.H. et al. 2008 Mol. Cell. Biol. 48 (15): 4851-4861 “How a cytokine is chaperoned through the secretory pathway by complexing with its own receptor: Lessons from interleukin-15
(IL-15) / IL-15 receptor alpha”.
ACKNOWLEDGEMENTS: This project has been partially funded by University of Valladolid (VA016A10-2), Health Institute Carlos III (PI10/01647) , Junta de Castilla y Leon (FPI,
Montalvillo E.) and University of Valladolid (FPI-UVa; Escudero-Hernández C., Martínez-Abad B.)